HU225762B1 - Robinia pseudoacacia lectin and its uses - Google Patents

Description

The present invention relates to the use of Robinia pseudoacacia (RPA) lectin in medicine and to an isolated Robinia pseudoacacia lectin homopolypeptide, to compositions containing Robinia pseudoacacia lectin, to the use of Robinia pseudoacacia in the manufacture of a medicament for the treatment and / or treatment of a disease. for the control, amelioration and / or treatment of a disease; and a diet or nutritional supplement containing Robinia pseudoacacia lectin.

Lectins are proteins or glycoproteins characterized by the ability to recognize specific glycoconjugate structures (Pusztai, A .: Plant Lectins Cambridge, Cambridge University Press (1991)). Some orally digested lectins are resistant to digestion in the stomach and reach intestine intact. In the intestine, lectins may bind to carbohydrate structures expressed on various intestinal mucosa cells (Pusztai, A., Ewan, S. W. B., Grant G., Peumans, W. J., Van Damme, E. J. N., Coates, Μ. E. and Bardocz, S., "Lectins and Lower Bacteria Modify the Glycosylation of the Gut Receptor in the Rat", Glycoconjugate Journal 12, 22-53 (1995)]. The consequences of attaching lectin to gut cells may be different. The biological responses, for example, depend on the dose administered. At high doses, renal bean lectin (PHA) causes reversible disorganization of the epithelial cell fluff. This is quickly recovered by endogenous processes. Similarly, in the presence of large amounts of PHA in the small intestine, an overgrowth of endogenous Escherichia coli occurs. In addition, lipid mobilization and glucose oxidation are increased and protein synthesis in skeletal muscle is reduced. However, at lower doses of PHA, an increased rate of cryptic cell production can be observed, leading to hyperplastic growth in the gut [Bardocz, S., Grant, G., Ewen, SWB, Duguld, TJ, Brown, D.S., Englyst, K. and Pusztai, A. : Reversible effect of Phytohemagglutinin on growth and metabolism of the rat gastrointestinal tract, Gut 37, 353-360 (1995)], but without the adverse effects that occur at higher doses.

Robinia pseudoacacia (White Acacia) is a legume tree species that is widespread in both North America and Central Europe. Robinia pseudoacacia produces lectins in the cortex, roots, tubers, bark, tree and leaves [Gietl, C. and Ziegler, H. Biochem. Physiol. Planzen 175, 58-66 (1080)], and in seeds [Van Damme, EJM, Barre, A., Rouge, P., Van Leuven, F., and Pneumaas, W., "The Seed Lectins of Black Locust ( Robinia pseudoacacia) are encoded by two genes which differ from the bark lectin genes, Plant Molecular Biology 29, 1197-1210 (1995)]. Although the exact function of lectins is unknown, it is believed that lectins can be used to store nitrogen during sleep periods [Yoshida, K., Baba, K., Yamamoto, N., and Tazaki, K.: Cloning of a lectin cDNA and seasonal changes in levels of lectin and its mRNA in the inner lobe of Robinia pseudoacacia, Plant Molecular Biology 25: 845-853 (1994)]. It is also known that lectin levels in the bark increase in winter. Due to the known toxicity of Robinia pseudoacacia in mammals [Nishiguchi, M., Yoshida, K., Sumizono, T. and Tazaki, T .: Studies on site-directed mutagenesis of carbohydrate binding properties of bark lectin from FEBS Letters 43 , 294-298 (1997)] it is also possible that lectin in the bark acts as a defense mechanism against winter herbivores such as rabbits.

Bark and seedlings of Robinia pseudoacacia have been characterized at both biochemical and molecular levels [Van Damme, E. J. M., Barre, A., Smeets, K., Torrekens, S., Van Leuven, F., Rouge, P. and Peumans, W.

J.: "The bark of Robinia pseudoacacia contains a complex mixture of lectins", Plant Physiology 107, 833-843 (1995); Van Damme, EJ M. "Barre, A., Rouge, P." Van Leuven, F., and Pneumaas, W.: "The Seed Lectins of the Black Locust (Robinia pseudoacacia) are Encoded by Two Genes That Differ From the Bark Lectin Genes. Plant Molecular Biology 29: 1197-1210 (1995). Root lectin has also been isolated and characterized (Duverger, E. and Delmotte, F.M., "Purification of lectins from Robinia pseudoacacia L. root-tips", Plant Science 123, 9-18 (1997)). Robinia pseudoacacia lectins (mainly nuclear ones) are used in many different ways, mainly as diagnostic and research tools. The specific carbohydrate-binding properties of RPA (N-acetyl-D-galactosamine) have been used to study the carbohydrate composition of biological tissue [Raedler, A., Boehle, A., Otto, U., and Raedler, E., "Differences of glycoconjugate exposed on hypernephroma and normal kidney cells (Journal of Urology 128: 1109-1113 (1982)). The mitogenic properties of RPA have also been used in in vitro assays to investigate the biology of isolated lymphocytes [Sabeur, G., Wantyghem, J. and Schuller, E., "Stimulation of 2 ', 3'-cyclic nucleotide 3'-phosphodiesterase in human lymphocytes. by Robinia pseudoacacia lectin ', Biochemie, 68, 581-585 (1986); Sharif, A., Brochier, J. and Bourillon, R., "Specific activation of human T lymphocytes by Robinia pseudoacacia seeds lectin", Cellular Immunology 31, 302-310 (1977). Banach et al. Have shown that intraperitoneal doses of RPA induced hepatotoxicity in mice, including a rapid reduction in hepatic glycogen content [Banach, M., Zaremba, S. "Sadouska, M.:" Disturbance of liver and muscle glycogen levels. in mice following administration of lectin extracted from Robinia pseudoacacia ', Folia Bioi. (Krakow, 31, 177-186 (1983)). Pusztai also confirmed that RPA is highly bound to rat intestinal wall and at high doses induces pancreatic growth, suggesting that RPS may damage the intestinal wall, promote coliform growth and induce pancreatitis.

Lectins derived from the bark of Robinia pseudoacacia can be characterized by a variety of techniques including various gel separations, DNA and protein sequencing techniques [Van Damme, EJM, Barre, A., Smeets, K., Torrekens, S., Van Leuven, F., Rouge, P. and Peumans, WJ: "The bark of Robinia pseudoa2

HU 225 762 Β1 cac / 'contains a complex mixture of lectins', Plant Physiology 107: 833-843 (1995)]. The natural cortical lectin eluting from one column consists of two lectins, one of which is RPbAI and RPbAII, which are purified together at an apparent molecular weight of 120 kDa. Analysis of RpbAI on SDS-PAGE gel shows that it consists of two subunits, polypeptide a and polypeptide b, having a molecular weight of 31.5 kDa and 29 kDa, respectively. RpaBI is a tetramer containing all possible combinations of polypeptide monomers a and b. Polypeptides a and b can be further characterized by cDNA cloning and sequencing using standard techniques. The predicted sequence of polypeptide a and b is shown in Figure 1.

RPbAII is a tetramer containing a single 26 kDa monomeric polypeptide (c subunit). The polypeptide c was further characterized, the cDNA was sequenced, and the predicted protein sequence is also shown in Figure 1.

Robinia psaudoacacia core contains two lectins, RPsAI and RPsAII, which are purified together at an apparent molecular weight of 120 kDa (Van Damme, EJM, Barre, A., Rouge, P., Van Leuven, F.S. and Pneumaas, W. : "Seed lectins of the black locust (Robinia pseudoacacia) are encoded by two genes which differ from the bark lectin genes" (Plant Molecular Biology 29, 1197-1210 (1995)). Analysis of both lectins by SDS-PAGE shows that each contains a single monomeric subunit (34 and 29 kDa). Maglectins were characterized by cDNA cloning and sequencing techniques and the predicted protein sequence is shown in Figure 2. Recent work with root lectins shows that two lectins (Duverger, E. and Delmotte, FM: "Purification of lectins from Robinia pseudoacacia L. root-tips", Plant Science 123, 9-18 (1997)). Root lectins appear to differ from seed and bark lectins,and dimeric molecules that can be graduated in 58 and 63 kDa, RprAI or RPrAII group. RprAI is a heterodimer consisting of 31 and 29 kDa subunits, RPrAII is a homodimer containing a single 30 kDa subunit.

Robinia pseudoacacia lectins have caused some confusion as to the exact molecular weight and subunit composition of each lectin. Wantyghem et al. Isolated two lectins in seeds, RPA 1 a dimer and RPA 3 a tetamer [Wantyghem, J., Goulut, C., Frenoy, J.P., Turpin, E. and Goussault, Y .: Purification and characterization of Robinia pseudoacacia seed lectins ”(new study), Biochem. J. 237: 483-489 (1986)]. However, in the same year, Fleischmann et al. Described the isolation of two tetrameric lectins, designated RPA 1 and RPA 2, with subunits of similar molecular weights [Fleischmann, G. and Rudiger, H., "Isolation, resolution and partial characterization of two Robinia pseudoacacia seed lectins" , Bioi. Chem. HoppeSeyler 367, 27-32 (1986)]. Thus, it appears that lectins exist in monomeric, dimeric, trimeric and tetrameric forms.

Mucosal membranes are composed of mucus cells and mucous membranes are wet membranes that delimit tubular structures and cavities in living organisms. Includes mucous membranes covering the intestines, mouth, nasal cavity, esophagus, stomach, lungs, small intestine, and colon (including rectum). The importance of mucus membranes in the viability of an organism has long been recognized. However, the ability to regulate the proliferation of mucous and non-mucus cells has not previously been recognized as providing an opportunity to address a number of physiological problems, including diseased and damaged cells and / or tissues. A number of disorders of mucosal cells are known, including conditions in which cell proliferation is accelerated, retarded, where mucus cells are damaged, or where the outer protective layer, i.e., mucus, is absent. Conditions associated with abnormal proliferation of mucus cells include skin cancers, psoriasis, colon irritation syndrome, inflammatory disease, and mucositis.

Mucositis is a painful and debilitating condition in which rapidly growing epithelial and mucosal cells are damaged and the outer mucous membrane either disappears and / or is not replaced rapidly enough. Mucositis can result in infection from microorganisms such as those found in the mouth or intestine. In this context, mucositis refers to intestinal lesions (which occur as damage to the mucous membrane after exposure). This condition is considered to be the main side effect of cancer treatment. The incidence and severity of mucositis may increase with newer stages of cancer therapy, and may ultimately affect compliance and survival. The solution provided by the present invention takes advantage of the metabolic effects and tissue protection properties of lectins on biological agents.

Diseased and damaged cells, which are unable to recover or regenerate fast enough, pose a serious and potentially life-threatening health risk. Metabolic diseases such as obesity, hyperglycemia, circulatory disease, stroke and gastrointestinal disease, including colon irritation syndrome and abdominal disease, are also problematic in maintaining normal cellular functions.

Cells and tissues can become ill and damaged for a variety of reasons. Cell-damaging agents include: chemotherapeutic agent (s), radiotherapy, chemical (s) (organic or inorganic), toxins, acid, alkali, radiation source, free radical, and many can cause damage through surgery or therapy, which is an attempt to treat an alternative, more problematic disease. Treating cancer is an excellent example of a cell and / or tissue damage in an organism as a side effect of another procedure. Treatment of cancer is primarily based on the use of chemotherapy or radiotherapy, either alone, in combination, or in combination with surgery. Chemotherapy uses a cytotoxic agent that directly damages it

EN 225 762 Β1 DNA of a target cell. If a sufficient dose of a cytotoxic agent is administered to a target tissue, such as a tumor, poor repair of DNA may result in the accumulation of DNA mutations, lesions and chromosomal aberrations, which eventually result in cell death. Radiotherapy uses radiation to either directly damage the DNA of a target cell or to exploit the potential of ionizing radiation to produce free radicals capable of breaking DNA strands [Steel, GG: "From targets to genes: a brief history of radiosensitivity, Phys. Med. 41: 205-222 (1996). Thus, chemotherapy or radiotherapy is a compromise in which we try to cause the most damage to the cancer cells by directing the radiation so that it does not irreparably destroy the normal tissue.

Although chemotherapy and radiotherapy are the most widely used treatment for cancer, the survival rate is limited by many different factors. The key factor is that the cytotoxic agent or radiation does not differentiate between normal and cancerous cells. In most cases, it is impossible to administer a sufficient dose of the cytotoxic agent or radiation to reliably destroy the cancerous tissue, as this may prove fatal to the patient. Common side effects of existing treatments include baldness, bone marrow suppression, nausea, vomiting and vomiting [Paulsen, F. "Hoffman, W., Kortmann, R. D., Porschen, R., and Bamberg, M.," Acute gastrointestinal Nebenwirkungen in dér Radio-oncologie - Was ist gesichert in dér Therapie ?, Strahlenther. Prime cost. 172, 53-56 (No. 2) (1996)]. In addition, there are many cases in which radiotherapy, particularly in the pelvic regions, has resulted in altered gastrointestinal function [Yeoh, E., Horowitz, M., Russo, A., Muecke, T., Ahmad, A., and Chatterton, B .: International Journal of Radiation Oncology, Biology & Physics 26: 229-237 (1993)] and, in the long term, intestinal damage requiring surgery [Van Halteren, Η. K., Gortzak, E., Taal, B.G., Helmerhorst, Th, J, M., Aleman, Β. Μ. P. Haart, A.A.M. and Zoetmulder, F.A.N., "Surgical Interventions for Complications Caused by Late Radiation Damage: A Retrospective Analysis, European Journal of Surgical Oncology 19, 336-341 (1993)].

There have been major breakthroughs in the last 10 years with the availability of hematopoietic growth factors. It is now possible to administer higher doses of cytotoxic agents and save tissues such as bone marrow and white blood cells by administration of recombinant growth factors such as granulocyte macrophage colony stimulating factor [Erkisis, M., Erkurt, E., Ozbarlas, S. , Burgut, R., Dórán, F., and Seyrek, E.: "The Use of Recombinant Human Granulocyte Colonystimulating Factor in Combination with Single or Fractionated Dosages of Isofamide and Doxorubicin in Patients with Soft Tissue Sarcoma," Journal of Chemotherapy 8, 224 -228 (1996)]. This approach allows for better prognosis and survival rates. While epidermal specific growth factors, such as epidermal growth factor, are known, the complex nature of gut growth regulation has made it difficult to develop effective bowel rescue procedures [Podolsky, D.

Q .: Regulation of Intestinal Epithelial Proliferation: A Few Answers (1993). Since no such growth factor is currently known in the gut, damage to the gastrointestinal tract by cytotoxic agents and irradiation has thus become a dose constraint.

The present invention solves these problems known in the art by providing and utilizing a novel and effective product that allows positive and controlled growth of the biological material or of the biological material.

Accordingly, the present invention relates to the use of Robinia pseudoacacia lectin for medical purposes. Its use in medicine is widespread. This includes proliferation of mucus cells, reduction and / or treatment of damage caused by cell damaging agents, or a combination of the two (or more). In this context, the term "reduction" means any effect that alleviates any injury or medical condition to any extent and includes prevention (prophylactic use). In this context, "treatment" provides any amelioration of a disorder, disease, syndrome, condition, pain, or a combination of two or more thereof. The treatment (or use of the compositions of the present invention) of the present invention may be carried out in a further "treatment" such as chemotherapy and / or radiotherapy.

Robinia pseudoacacia lectin is particularly useful in controlling mucosal cell proliferation since it is involved in the reduction and / or treatment of mucositis, including intestinal lesions. Controlling the proliferation of mucus cells is particularly useful in alleviating and / or treating damage caused by a cell damaging agent. Radiotherapy, chemotherapy, or a combination thereof may be typical cell-damaging agents in all aspects of the present invention. In the present invention, the terms "radiation" and "radiotherapy" are used interchangeably, as a source of radiation (radiation), which may or may not be used as a therapeutic technique.

The invention is particularly useful for reducing and / or treating damage to mammalian cells, particularly human tissues. The present invention is particularly important in human tissue, as radiation therapy and / or chemotherapy play an important role in the treatment of cancer. However, the present invention can also be applied to other animals and biological materials, particularly in the treatment of animals, including farm animals and domestic animals.

The use of Robinia pseudoacacia in medicine applies to all cells and / or tissues, including cells and tissues within the whole body and outside the whole body, including isolated cells and tissues. Applies to all biological

EN 225 762 Β1 including whole bodies and parts thereof.

In addition, the invention relates to any material which, intentionally or unintentionally, comes into contact with a cell-damaging agent.

The invention is particularly useful for biological material which is particularly sensitive to cell damaging agents such as radiotherapeutic or chemotherapeutic agents. Such biological agents include the mucous membranes, particularly the intestine, mouth, nasal cavity, esophagus, stomach, lungs, small intestine, colon (including colon and rectum), epithelial tissue (e.g. and any other mucosal cells and / or tissues and non-mucous cells and tissues such as bone marrow, spleen, all hematopoietic cells and tissues, blood tissue, thymus, hair follicles, eye tissue, reproductive system, testis / prostate tissue, or a combination of two or more of these. The present invention is particularly useful for mucous membranes, which are tissues that cover the body cavities, including permanently wet tissues that cover the eyes. The sensitivity of cells and tissues to the deleterious effects of a cell damaging agent can be linked to their metabolic state. The effect of Robinia pseudoacacia lectin growth factor, particularly in the intestine (and especially in the small intestine), is known to be important in prophylactic and / or therapeutic effects during and / or after administration of cell damaging agents.

Growth is particularly well controlled by Robinia pseudoacacia lectin during and / or after radiotherapy, chemotherapy or a combination of the two. The invention was made possible by the discovery of the protective and restorative capabilities of Robinia pseudoacacia lectin.

Mucous cells form the mucous membrane (the wet membrane covering various tubular structures and cavities). Many of these form a protective layer between the external environment and the internal organs of an animal. Mucosal cells / tissues include the intestine, mouth, nasal cavity, esophagus, stomach, lungs, small intestine, colon, epithelial tissue, and any other mucosal cells and / or tissues. The present invention relates to all of the aforementioned cells and tissues. The present invention also relates to bone marrow, spleen, all hematopoietic cells and tissues, blood tissue, thymus, hair follicles, ocular tissue, reproductive organs, testis / prostate tissue, or a combination of two or more thereof.

According to the present invention, Robinia pseudoacacia lectin can be used in medicine in combination with a cell protective agent. The cell protection agent is preferably a radiosensitizer, a chemoprotectant (including free radical scavengers), a growth factor, or a combination of two or more thereof. Among the cell protective agents we prefer the following: Radiosensitizers - Vitamin K mimetics such as Synkavit or Menadione, Gadolinium texaphyrin or iobengane ([[(3-iodo-13311) -phenyl] -methyl] -guanidine: chemoprotectants - Sulcraphate, cysteine, cysteamine, Ethyol, balazipone or dosmalfate; free radical scavenger - WR 3689 (2 - [[3-methylamino) propyl] amino] ethanediol dihydrogen phosphate ester, AD 20 (2 - [[2-methoxyphenyl) acetyl] amino] -2 -propenoic acid or nitroxide antioxidant; growth factors granulocyte colony stimulating factor (G-CSF) granulocytamacrophage colony stimulating factor (GM-CSF), erythropoietin (EPO), epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor (TGFa and β), any interleuk including IL-11 and IL-15, insulin-like growth factor (IGF), nerve growth factor (NGF), platelet-derived growth factor (PDGR), Bombesin, Relaxin, Calcitonin, Whey Milk Growth Factor (CDGF), amlexanox or amoxanox, protegrin, pilocarpine hydrochloride, stem cell factor (STF), thrombopoietin, iron factor (SF), any interferon including interferon or any cytokine.

In addition to the cell protective agents, the lectin may be used in combination with one or more other compounds / agents (preferably drugs). More specifically, antimicrobial agents are included. Such agents are used to combat secondary infections, such as secondary infections associated with mucositis.

The therapeutic use of Robinia pseudoacacia lectin in medicine is particularly useful in relation to cells of the digestive tract (both for reduction and repair of damage and for metabolic disease). The aim of the control may be to increase the function and / or length of the digestive tract or to regulate the nature and / or density of cell surface glycoproteins expressed on the digestive cells. Other applications related to the regulation of mucosal cells include, for example, the reduction and / or treatment of intestinal disorders, such as inflammatory bowel disease and intestinal irritation syndrome, and the reduction and / or treatment of intestinal lesions, and the repair and replacement of mucosal cells. before, during or after chemotherapy or a combination of these.

Applications of these control properties include:

(a) intestinal proliferation, which is the functional intestinal tract and

leading to an increase in length, which may be a useful therapy where bowel function is impaired, such as during surgery or injury, and / or (b) controlling the rate of bowel turnover, which allows the nature and density of expressed surface glycoconjugates to be controlled, they become more complex as the cell ages. Because glycoconjugates affect certain bowel properties, such as their propensity to bacterial attachment, administration of lectin can be considered as a therapeutic or prophylactic regulation of these properties.

Specifically, control of cell proliferation can be used to achieve increased nutritional capacity of the small intestine and / or to regulate glycoconjugation.

EN 225 762 Β1 gut expression. These effects are not necessarily medical disorders and may be only cosmetic or functional disorders below or above the appropriate medical level.

These effects may be useful in the use of Robinia pseudoacacla lectin, in the reduction and / or treatment of a metabolic disorder.

Metabolic disorders include any disorder related to and / or resulting from the metabolism of the body, more particularly obesity and obesity-related disorders such as hyperglycaemia (type II diabetes), cardiovascular, stroke, gastrointestinal and conditions associated with the gastrointestinal system. A metabolic disorder may require regulation of mucosal cell growth, or control of mucosal cell growth may be independent of the metabolic disorder. This includes non-medical weight loss, which can be simply cosmetic.

According to the present invention, the higher the concentration of lectin, the faster the reduction and / or treatment of any disease or other non-medical syndrome. However, a number of factors may influence the preferred concentration of lectin dose as discussed below.

According to the present invention, Robinia pseudoacacia lectin may be in its natural form or purified, may be chemically synthesized or may be produced by recombinant technology. Lectin can be modified or its derivative (naturally occurring or synthetic). Methods for producing lectin are well known in the art, both by isolation from natural sources and by recombinant technology (Pusztai and Palmer (1977); Carvalho (1993); U.S. Patent No. 4,889,842]. Preferably, the lectins of the invention are functionally equivalent to the "originally isolated Robinia pseudoacacia lectins. The functionality of any Robinia pseudoacacia lectin can be determined by its ability to bind specific glycoconjugate structures. Most preferably, the polypeptide is a Robinia pseudoacacia lectin of the present invention when equivalent (or a lectin variant as defined below), i.e. 40%, preferably 50%, preferably 60%, more preferably 70%, and more preferably 80% or more, at the same amino acid level or DNA sequence level with any known Robinia pseudoacacia lectin, such as the lectins shown in Figures 1 or 2.

The term "lectin variant" as used herein refers to a polypeptide which is substantially homologous to natural lectin, but whose amino acid sequence is different from that of natural lectin (human, rodent, or other mammalian species) due to one or more deletions, insertions or substitutions. Preferably, the variant amino acid sequence is at least 80% identical to the natural lectin amino acid sequence, more preferably at least 90% identical. For example, the percent identity can be determined by comparing the sequence information obtained with the GAP computer program (version 6.0) described by Devereux et al., Available from the University of Wisconsin Genetics Computer Group (UWGCG) [Devereux et al., Nucleic Acids Research 12: 387 (1984)]. The GAP program uses the Needleman and Wunsch fitting method (Needleman and Wunsch, 1970, Journal of Molecular Biology 48: 443), following a review by Smith and Waterman (Smith and Waterman, Adv. Appl. Math. 2, 482 (1981)].

Since lectins are proteins, it is clear that many parameters such as heat, acid, alkali, etc. may be the subject of destabilization / denaturation. Because the protein properties of the lectins used in the present invention are important, it is important that the lectins are not completely destroyed or denatured prior to or during use (e.g., under strongly acidic conditions of the stomach and mildly alkaline conditions of the lower intestine). . Accordingly, in order to use Robinia pseudoacacia lectins in the present invention, it may be necessary to first characterize them in terms of their processing and / or administration. Such characterization is standard technology and can be easily accomplished by one skilled in the art. The concentration required for the use of lectin in the present invention varies depending on whether the lectin is denatured or destabilized in any way.

Concentrations of Robinia pseudoacacia lectins according to the present invention are determined by their natural properties and are not reduced by denaturation or destabilization. Thus, the concentrations of the lectins of the present invention are not absolute but reflect the activity of the lectin. Accordingly, for example, a composition having a lectin which has a half activity but which is present in a double concentration is the same composition having only half the amount of lectin but having no activity. In addition, the activity of any lectin may be increased or decreased by modification, for example, by recombinant production and / or by production of truncated mutants that have increased activity. The same considerations apply between lectin concentration and activity, even for lectins that have increased activity. The present invention also relates to all modified Robinia pseudoacacia lectin or derivatives thereof, including enhanced or reduced activity, and includes, for example, truncated lectin units and / or differently glycosylated versions with full or modified activity. Also included are protein sequences containing one or more of the critical amino acids involved in any lectin biological activity (preferably all such amino acids and total biological activity). For example, the RPA polypeptide is b sec6

The active regions of HU 225 762 Β1 were described by Nishiguchi et al. And contain the following amino acids (with position designation): aspartate: 87, phenylalanine: 130, threonine:

215, leucine: 217, serine: 218.

The Robinia pseudoacacia lectin of the present invention may be derived from any portion of the Robinia pseudoacacia plant, or may be equivalent to a lectin derived from any portion of the Robinia pseudoacacia plant, including bark, seeds, root, tuber, bark or tree, or a combination of two or more of these. According to the present invention, Robinia pseudoacacia lectin may comprise one or more polypeptide subunits. The polypeptide subunit may be any of the subunits known in the art (such as RPbAI (a or b), RPbAII (c), RpsAI or RPsAII, RprAI or RPrAII), or an equivalent thereof, or a polypeptide subunit not yet defined. Preferably, the Robinia pseudoacacia lectin of the present invention may comprise a monomer, dimer, trimer or tetramer of subunits, or a combination of two or more thereof. This makes it possible to mimic a naturally occurring polypeptide cluster. The monomer, dimer, trimer, tetramer may be the same polymeric subunit or may be a combination of more than one type of polypeptide subunit. Preferably, the tetramer comprises four bark polypeptide a subunit, four bark polypeptide b subunit, or an intermediate mixture of a and b subunits. The lectin polypeptides may contain or correspond to one or more of the amino acid sequences shown in Figures 1 or 2, preferably 40, 50, 60, 70, 80, 90, 100% homologous thereto (as the lectin variants described above).

The present invention further provides an isolated Robinia pseudoacacia lectin comprising a homotetramer, homotrimer, homodimer or a single subunit, preferably purified at 60% or better. In this context, the term "purified" means that the material is substantially free of impurities. Robinia pseudoacacia lectins purified to such an extent are preferred for use in pharmaceutical compositions and for use in medicine. Robinia pseudoacacia lectin can preferably be purified in 70%, 80%, 90% or more, most preferably in 95%, 99% or 100%. Advantageous aspects of the present invention apply to all embodiments of the invention.

The present invention further relates to the use of Robinia pseudoacacia lectin in the manufacture of a medicament for use in the control of mucosal cell proliferation, for the reduction and / or treatment of damage caused by a cell damaging agent, for the reduction and / or treatment of a metabolic disorder. The preferred properties are the same as those described above.

The patient is believed to be in need of such medication who will receive, receive, or have received radiotherapy and / or chemotherapy, especially if he or she has cancer.

The Robinia pseudoacacia lectin of the present invention may also be used as a dietary supplement. A diet or dietary supplement may contribute to non-medical weight loss. The diet or dietary supplement can be used in all animals, including humans.

Preferably, the diet or dietary supplement is used for 2 to 5 days or for a long (infinitely long) period. The diet or dietary supplement preferably contains 0.3 g, preferably 0.2 g total lectin per kg body weight. At least a portion of the lectin must be Robinia pseudoacacia lectin (preferably 1-100% of the dietary or supplemental lectin content). Robinia pseudoacacia lectin may be any lectin according to the first subject of the present invention, or a combination of any two or more of the foregoing. Such a diet or dietary supplement may be useful in medicine, particularly in the control of mucosal cell proliferation, in the reduction and / or treatment of damage caused by a cell-damaging agent, in the reduction and / or treatment of a metabolic disorder, or in combinations of two or more thereof.

The diet and / or dietary supplement is particularly useful if it is followed by a period of good quality nutrition. A high quality diet is defined as a diet that provides all the essential proteins, fats, carbohydrates, minerals, and vitamins needed for the normal growth of an animal. A good quality diet contains the essential components in the optimum proportion. A good quality diet should not contain any component that slows or inhibits the growth or development of the animal. A preferred feature of a good quality diet is that the food consumed is converted positively to lean body weight.

Preferably, a high quality diet follows the lectin diet and / or dietary supplement immediately or shortly (up to two days). A good quality diet is most beneficial when it lasts for up to seven days, preferably up to 5 days. Prolonged use of the diet and / or dietary supplement (more than one month, preferably more than one year) may be useful in the treatment of metabolic disorders and cosmetic weight loss.

When used in combination with a cell-damaging agent (though not necessarily at the same time as administration), it is best to limit the lectin-containing portion of the diet to a maximum of 5 days, as 48-72 hours are not sufficient to complete a complete cycle of intestinal turnover. the advantages of food absorption and application rate are obtained in the diet portion of the diet control. It is best to limit the portion of the cycle that provides good quality nutrition to about 5 days in order to maximize the efficiency of the food conversion.

Preferably, the diet and / or dietary supplement contained in a feeding period with Robinia pseudoacacia lectin is followed by a high quality diet.

EN 225 762 Β1 feeding period, it is repeated at least twice to establish a cyclic diet. The cycles are repeated up to twenty times, preferably up to ten times, and most preferably six times.

The described cyclic diet temporarily increases the efficiency of food intake mediated by administration of Robinia pseudoacacia lectin. As a result, food-dependent situations can be planned by organizing lectin-containing and lectin-free diets in a cycle. For example, with the right food timing, an athlete can optimize his performance for the main races.

The diet and / or dietary supplement is particularly useful before or after radiation and / or chemotherapy treatments. In such situations, diets may be even more effective when used in combination with a cell protective agent, such as those described in the first aspect of the present invention.

For each subject of the invention, which may play a role in medicine or be administered in biological tissue, the dosage range employed will ultimately be determined by the attending physician, who will consider factors such as the type, age, weight of lectin or lectins used, the severity of the symptoms and / or the treatment to be applied, the route of administration, the adverse side effects and / or other contraindications. Specific dose ranges can be determined by standard designed clinical trials, with complete follow-up of changes in patient status and recovery. In such assays, increasing dose schedules can be employed, using a low percentage of the maximum tolerated dose in animals as a starting dose in humans. Dose ranges can be derived from the results obtained in the experimental section of this text and from the human extrapolated ranges below.

A suitable upper limit appears to be a concentration of 0.3 g lectin / kg body weight per day. A concentration of 0.3 g per kg of body weight per day will certainly cause gastrointestinal problems in a patient, but these symptoms are acceptable on the basis that it increases the patient's chances of survival, for example from cancer. A suitable lower limit appears to be a concentration of 0.0001 mg (0.1 pg) per kg body weight per day of lectin. Preferred intermediate concentrations include 0.2, 0.15, and 0.05 g of lectin per kg body weight per day, followed by lectin at 1 mg, 0.5 mg, 0.1 mg, 0.01 mg and Concentration of 0.005 mg / kg bw / day.

The medicament of the present invention may be used to provide the desired concentration of lectin in one or more "doses" per day (the term "revenue" is intended to mean all modes of administration and includes a unit dose). Some of the objects of the present invention may be suitable for a single intake of a high concentration of lectin, while other objects may be for a multiple intake, evenly or unevenly distributed over the same period, but with a lower concentration of lectin per revenue.

The use of lectin in any of the medicaments of the present invention (including the preparation of the medicaments) may be in combination with suitable pharmaceutical carriers and / or excipients. Such carriers and excipients are well known in the art, Handbook of Pharmaceutical Excipients, 2nd Edition, edited by A. Wade / P. J. Weller, The Pharmaceutical Press, American Pharmaceutical Association (1994). Particularly useful according to the present invention are formulations and / or medicaments which are formulated for intestinal absorption such as mouthwash, cream, ointment or capsule.

Preferably, the medicament prepared according to any of the present invention is administered orally (orally), nasally (in the form of a nasal spray) or rectally (for the simple administration of one or more portions of the gastrointestinal tract), although parenteral or intravenous administration may be employed.

The present invention is illustrated by the following examples.

Examples

Production of lectin from the bark of Robinia pseudoacacia

The lectin in the cortex of Robinia pseudoacacia can be prepared essentially by the methods published by Van Damme et al., From partially purified bark extract immobilized on fetuinone (Van Damme, EJM, Barre, A., Smeets, K., Torrekens, S., Van Leuven, F., Rouge, P. and Peumans, WJ: "The bark of Robinia pseudoacacia contains a complex mixture of lectins", Plant Physiology 107: 833-843 (1995).

Bark tissue was homogenized in 20 mM acetic acid containing 200 mg / L ascorbic acid using a blender. The homogenate is pressed through a gauze, adjusted to pH 5.0 (with 1 M sodium hydroxide) and centrifuged at 10,000 g for 10 minutes. The resulting supernatant was filtered through glass wool to remove any suspended particles. After addition of 1.5 g / l CaCl ₂ , the pH of the extract was adjusted to 9.0 (with 1 M sodium hydroxide) and kept overnight at 4 ° C. The resulting precipitate was removed by centrifugation and the pH of the clear extract was adjusted to 3.8 with 1 M hydrochloric acid. After standing overnight at 4 ° C, the extract was purified by centrifugation, adjusted to pH 7.0 with 1 M sodium hydroxide, and then adjusted to 1 M ammonium sulfate by addition of a solid salt. The solution is then degassed and centrifuged again. The purified supernatant was loaded onto a fetuin agarose column equilibrated with 1 M ammonium sulfate, pH 7.0. After passing through the extract, the column was washed with ammonium sulfate until the A ₂₈₀ value was less than 0.01. The lectin is then desorbed with 20 mM diaminopropane (pH 11). The pH of the desorbed lectin was adjusted to 7.0 with 1 M hydrochloric acid and the solid salt was adjusted to 1 M ammonium sulfate. After centrifugation for 20 minutes at 15,000 g, the supernatant was loaded on a phenyl-Sepharose column equilibrated with 1M ammonium sulfate. The impurities are removed by washing with 1 mol / L ammonium sulfate and then the lectin is washed with 1 mol / L ammonium sulfate.

EN 225 762 Β1 with a linear gradient of water and water. Fractions were collected and their A ₂₈₀ values were determined to identify those containing lectin.

It is known that bark lectin is produced by Robinia pseudoacacia in response to a reduction in the length of the day, such as the onset of winter. When winter bark material is used, lectin production reaches 10 mg / g.

Preparation of lectin isotypes of A ₄ and B ₄ Robinia pseudoacacia bark

Homotetramer A ₄ and B ₄ isotypes can be prepared by step gradient ion exchange chromatography. A 100 mL Hyper-D S column (BioSepra) was equilibrated with five column volumes of 50 M sodium acetate solution (pH 4.75). Freeze-dried Robinia pseudoacacia lectin (prepared according to the previous preparation step) is dissolved in acetate buffer (50 mM sodium acetate, pH 4.75). The purified supernatant was loaded onto the column and the column washed with acetate buffer at a flow rate of 3 ml / min. A gradient gradient was prepared using 50 mM sodium acetate (pH 4.75) and sodium chloride. Sodium chloride was obtained in increasing concentrations as gradients of 5, 7, 9, 12 and 100%. The lectin-containing fractions were selected on the basis of absorbance at 280 nm. The tetramer A ₄ elutes in the 5-7% salt fraction and the tetramer B ₄ elutes in the 9-12% salt fraction.

The fractions containing pure A ₄ and B _{4 were} analyzed by conventional polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis.

Characterization of lectins from Robinia pseudoacacia bark

Lectins from Robinia pseudoacacia bark can be further characterized by ion exchange chromatography, gel filtration and in vitro cellular agglutination assays. These methods are known in the art and are described by Van Damme et al., Van Damme, J. J., Barre, A., Smeets, K., Torrekens, S., Van Leuven, F., Rouge, P., and Peumans, WJ. The bark of Robinia pseudoacacia contains a complex mix of lectins', Plant Physiology 107: 833-843 (1995); Van Damme, EJM, Barre, A., Rouge, P., Van Leuven, F., and Pneumaas, W.: "The Seed Lectins of the Black Locust (Robinia pseudoacacia) Are Encoded by Two Genes That Differ From the Bark Lectin Genes. Plant Molecular Biology 29: 1197-1210 (1995).

Animal Husbandry

Thirty-five young male Hooded Lister (Rowett Strains) rats (19 days old, 30-40 g) were housed individually in lattice cages. The animals were fed a high quality semi-synthetic diet containing 10% lactalbumin protein (Table 1).

Table 1. Nutritional composition

g / kg feed corn starch 373 potato starch 100

g / kg feed glucose 150 Corn oil 150 vitamin Mixture 50 Mineral mixture 50 lactalbumin 127 silicic 0.4

Mixtures of vitamins and minerals are described in Carvalho, A. F. F. U. de: "Dietary Kidney Bean Lectins Affect Insulin Levels, Change Gene Expression and Modulate Metabolism", PhD Thesis, University of Aberdeen (1992).

The ability of orally ingested Robinia pseudoacacia lectins to protect rats from high-dose chemotherapy treatment, more specifically its intestinal tissue protection effect, was fed in five groups, each containing 5 rats, in a standard diet containing lactalbumin (Table 1). One group of rats was fed with 8 g / day of standard feed for three days and then injected with 5-FU at 150 mg / kg body weight (Table 2).

Table 2. Nutrition protocol

Treatment Nutrition protocol 1 LA 3 days, 5-FU injection, LA 6 days 2 25 mg / kg RPA for 3 days, 5-FU injection, LA 6 days 3 50 mg / kg RPA for 3 days, 5-FU injection, LA 6 days 4 100 mg / kg RPA for 3 days, 5-FU injection, LA 6 days 5 100 mg / kg RPA for 3 days, LA for 6 days

Explanation

LA = lactalbumin

RPA = Ro / n / a pseudoacacia lectin

5-FU = 5-Fluorouracil

The rats were fed in pairs for the first three days and then fed ad libitum after 5-FU injection.

Three groups of animals were dosed with 25, 50, 100 mg / kg of Robinia pseudoacacia lectin (RPA) via gastric tube for three days daily, and the next day injected with 150 mg / kg of 5-FU. The other groups of animals receive 100 mg / kg Robinia pseudoacacia lectin in advance and then return to the lactalbumin diet without injection of 5-FU. After injection with 5FU, the animals were returned to the lactalbumin-containing diet, ad libitum. Seven days after the start of the experiment, the animals are sacrificed, the carcass is dissected and the weight of the small intestine, fasting, and colon is recorded. The results are shown in Table 3.

HU 225 762 Β1

Table 3

Effect of three-day pre-dosing of Robinia pseudoacacia lectin on small, intestine, and colon weight (g)

Treatment dose range Small intestine (g) Hunger (g) Hip (g) 1 LA + 5-FU 5.62 1.0 1.0 2 25 mg / kg RPA + 5-FU 5.41 1.03 0.98 3 50 mg / kg RPA + 5-FU 5.74 1.12 1.08 4 100 mg / kg RPA + 5-FU 6.33 1.27 1.12 5 100 mg / kg RPA 6.99 1.27 1.17

The results show an increase in the wet weight of all tissues tested by increasing the amount of Robinia pseudoacacia lectin. Thus, pre-administration of Robinia pseudo doacacia lectin can protect the small intestine, the hunger and the iliac from damage by 5-FU. Pretreatment with Robinia pseudoacacia lectin 100 mg / kg (treatment # 4) gave the best tissue protection effect compared to animals receiving lactalbumin alone (treatment # 1).

Animals treated with 100 mg / kg Robinia pseudoacacia lectin and treated with 5-FU (treatment # 4) had a wet weight of small intestine, fasting, and hip that was treated with animals treated with 100 mg / kg Robinia pseudoacacia lectin alone (treatment # 5).

Visual evaluation of the small intestine, the intestine, and the colon indicates that animals treated with 5-FU only (treatment # 1) have very little or no mucosal layer on the intestinal tissue. However, animals treated with 5-FU and Robinia pseudoacacia lectin have normal intestinal tissue with normal mucous membrane.

This example demonstrates that oral administration of Robinia pseudoacacia lectin prior to 5-FU injection provides significant tissue protection in the small intestine, midgut, and colon.

Orally administered Robinia pseudoacacia lectin

Ability of ₄ and B ₄ homotetramers to protect rats from high dose chemotherapy, more specifically, intestinal tissue protection Five groups of 5 rats each were fed a standard diet containing lactalbumin (Table 1) and following the following nutrition protocol (Table 4).

Table 4. Nutrition protocol

Treatment Nutrition protocol 1 LA 7 days 2 LA 3 days, 5-FU injection, LA 4 days 3 200 mg / kg ₄ homotetramer 3 days, 5FU injection, LA 4 days 4 200 mg / kg B ₄ homotetramer for 3 days, 5FU injection, LA for 4 days

One group of animals (treatment 1) was fed a standard diet throughout. The second group receives saline through the gastric tube for three days daily and then injected with 150 mg / kg body weight 5-FU (treatment # 2). The remaining two groups of animals (Treatment Nos. 3 and 4) were fed through a gastric tube with Robinia pseudoacacia lectin homotetramer A ₄ or B _{4 for} three days and then injected with 5FU of 150 mg / kg body weight. After the 5-FU injection, treatment groups 2-4 were returned to standard diet for four days, ad lib. Seven days after the start of the experiment, 40 animals are sacrificed, the carcasses are dissected and the dry weight of the small intestine, fasting and colon is recorded. The results are shown in Table 5.

Table 5

Effect of three days pretreatment of Robinia pseudoacacia lectin A ₄ or B ₄ on dry weight of small intestine, jejunum and ileum (mg)

Treatment dose range Small intestine (mg) Intestine (mg) Hip (mg) 1 LA 2531 519 415 2 LA + 5-FU 1789 336 405 3 200 mg / kg A ₄ 2597 632 421 4 200 mg / kg B ₄ 2093 439 419

Dry weights were normalized to 100 dry weights.

The results show that treatment with 5-FU has a great effect on the small intestine and major airway in the colon. In animals treated with 5-FU, the dry weight of tissue was significantly reduced compared to untreated control (treatments 1 and 2). However, both A ₄ and B _{4 were} performed on a lectin homotetramer

Pre-treatment with 225 762 Β1 resulted in a significant tissue protection effect (treatments 3 and 4). Specifically, the tissue dry weight of animals treated with A ₄ homotetramer lectin is essentially the same as that of untreated animals.

Visual inspection of the small intestine, the intestine and the iliac gland shows that intestinal mucosa is severely damaged in animals treated with only 5-FU. However, animals treated with A ₄ or B ₄ homotetramer lectin have essentially normal mucous membranes.

This example demonstrates that both A ₄ and B ₄ homotetramer Robinía pseudoacacia lectin can provide significant intestinal protection when pretreated before 5-FU injection.

Claims (22)

PATENT CLAIMS Use of Robinía pseudoacacia lectin for the manufacture of a medicament for the control of mucosal cell proliferation, for the reduction and / or treatment of damage caused by cell damaging agents, or for combinations thereof. The use of Robinía pseudoacacia lectin according to claim 1, wherein the medicament is administered to a patient who is undergoing or has undergone chemotherapy treatment, or has undergone such treatment, especially where the patient is suffering from cancer. The use of Robinía pseudoacacia lectin according to claim 1 or 2, wherein the use is for the reduction and / or treatment of mucositis or intestinal lesions. The use of Robinía pseudoacacia lectin according to claim 1, wherein the use is for the reduction and / or treatment of intestinal disorders. Use according to claim 4, wherein the intestinal disorder is inflammatory bowel disease or irritable bowel syndrome. 6. The use according to any one of claims 1 to 4, wherein the Robinía pseudoacacia lectin comprises bark, seed, root, root tuber, bark, tree or leaf lectin or a combination of two or more thereof. 7. The use according to any one of claims 1 to 4, wherein the lectin unit (s) comprises a monomer, dimer, trimer or tetramer, or a combination of two or more thereof. The use of claim 7, wherein the lectin comprises a homopolypeptide. 9. The use according to any one of claims 1 to 6, wherein the lectin is a homotetramer of a or b crustal units. 10. The use according to any one of claims 1 to 4, wherein the lectin is a homotetramer, homotrimer, homodimer, or a single subunit or a combination thereof purified to a level of 60% or more, optionally a or b crustal units, or wherein the lectin is a homologue. The composition of claim 1 is present. 11. The use according to any one of claims 1 to 4 for use in mammalian cells and / or tissues, in particular in human cells and / or tissues. Use according to claim 11, wherein the cell and / or tissue is a mucosal cell, including a mucosa. 13. Robinía pseudoacacia lectin for medical use, which lectin consists of a homotetramer, homotrimer, homodimer or single subunit of the crustal unit, or a combination of two or more thereof. 14. Robinía pseudoacacia lectin for pharmaceutical use, which lectin consists of a homotetramer, homotrimer, homodimer or a single subunit of the crustal unit, or a combination of two or more thereof. Robinía pseudoacacia lectin according to claim 13 or 14, for use in the treatment of a patient, wherein the patient is undergoing or undergoing chemotherapy, in particular where the patient is suffering from cancer. 16. Robinia pseudoacacia lectin according to any one of claims 1 to 3, for use in controlling mucosal cell proliferation, reducing and / or treating damage caused by cell damaging agents, or a combination thereof. 17. Robinía pseudoacacia lectin according to claim 16, for use in the reduction and / or treatment of mucositis or intestinal lesions. 18. Robinía pseudoacacia lectin according to claim 16 for use in reducing and / or treating an intestinal disorder. The Robinía pseudoacacia lectin of claim 18, wherein the intestinal disorder is inflammatory bowel disease or irritable bowel syndrome. 20. A 13-19. Robinia pseudoacacia lectin according to any one of claims 1 to 3 for mammalian cells and / or tissues, in particular human cells and / or tissues. The Robinía pseudoacacia lectin of claim 20, wherein the cell and / or tissue is a mucosal cell, including a mucosa. A combination of Robinía pseudoacacia lectin and a cytoprotective agent for use in medicine.